Non-A non-B hepatitis is an infectious disease which is caused by a masked virus other than A and B hepatitis viruses, but it is not easy to identify the virus because an amount of the virus-specific antigens is very small in a patient's body as well as of anti-virus antibodies. Accordingly, diagnosis of non-A non-B hepatitis has been made serologically by the well-known "diagnosis by exclusion" method wherein increase in the levels of alanine aminotransferase and aspartate aminotransferase is determined for a serum from a patient to make a diagnosis whether or not the hepatitis belongs to any of hepatitis A, hepatitis B, hepatitis D and other hepatitis symptoms due to the known hepatopathy-causing viruses such as CMV, EBV, etc, and if the result of diagnosis are not applicable to them, then a case is identified as non-A non-B hepatitis. It, however, is difficult to diagnose clinically as being non-A non-B hepatitis by such a method because there is no correlation between ALT value and non-A non-B hepatitis. Also, the lack of trustworthy means for the diagnosis is a serious problem, whereby a secondary infection with the non-A non-B hepatitis virus which may be caused by transfusing blood, especially, from a non-A non-B hepatitis virus-carrying healthy carrier into a person can hardly be prevented. Therefore, it is considered that the non-A non-B hepatitis occupies more than 90% of hepatitis cases caused by blood transfusion, with a total of about one million patients per one year.
In order to improve such situation and to raise a diagnostic accuracy of non-A non-B hepatitis, Alter's panel in which a standard serum is used has been developed by Alter at al at the NIH. Diagnostic materials which can pass the Alter's panel have been obtained by Arima et al [JIKKEN IGAKU (Japan), 7 (2), 196-201 (1989)] and by M. Houghton et al (WO 89/04669, PCT/JP90/500880) of Chiron Corp. almost simultaneously. Arima et al have screened the sera from hepatitis patients using .lambda.gt11 (a protein expression vector) which is derived from viral RNA from a non-A non-B hepatitis patient's serum. Also, Chiron Corp. have inoculated the patient's blood plasma into a chimpanzee to develop a chronic hepatitis, blood plasma being obtained from the diseased animal which possesses the anti-virus antibodies with high titer, and then have screened in the same way as Arima et al. Chiron Corp.'s group has also succeed in cloning almost the whole portion of the gene of a hepatitis C virus (HCV, designated by Chiron Corp. ) and developed a kit for diagnosis which comprises an antigen protein obtained by expressing a part of the HCV gene.
In spite of such an effort, however, factors of this disease, even their numbers, have not yet been elucidated to the full.
As described above, the two materials which can pass the Alter's panel has certainly lead to a new technique of diagnosis replaced by said "diagnosis by exclusion", but screening patient's sera separately with the materials gives no results to be satisfied because both the materials from Arima et al and Chiron Corp. react with patient's sera in low positive ratios of about 60 to 80% and about 50 to 70%, respectively. In other words, in some cases, these materials would not react with sera from the patients who have been diagnosed clinically as non-A non-B hepatitis. A virus commonly have a function to cause mutation in their host cells for their surval, and thus the viral genes isolated from American patients by Chiron Corp. had been possibly mutated into various forms acclimated to the chimpanzee as an infection intermediate.
Accordingly, a great demand has been directed to a large scale preparation of the reactive antigens which are capable of probing the non-A non-B hepatitis patients or carriers, therefor it will be necessary to construct effective cDNA clones through the isolation and purification of variously mutated vital RNA from many non-A non-B hepatitis patients.
In addition, in the case of sera which have failed in a trustworthy diagnosis using an antibody detection system, or of sera which are collected immediately after infection and in which antibody titers do not yet raise, a gene amplification method (PCR method) may be useful for the confirmation of the disease because it can detect a trace amount of vital genes. Also, it is possible to clone the genes efficiently by the PCR method. However, since the PCR method is carried out using primers which are synthesized from a known gene sequence, it is not always possible to detect a gene of the non-A non-B hepatitis virus in a patient's fluid using a primer(s) which can be constructed on the basis of the HCV gene sequences determined by Chiron Corp., if a difference in mutation between said HCV gene of Chiron Corp. and said patient-carried vital gene is significant.
In consequence, to detect efficiently infection with the non-A non-B hepatitis virus, it is necessary to prepare at least one primer capable of detecting the viral gene with a high specificity. Such a purpose may be accomplished by isolating a great number of cDNA clones, synthesizing primers from relatively preserved regions among their gene sequences, and subjecting the primers obtained to screening through the PCR method.